Sorting cell/nuclear suspensions on basis of DNA ploidy

Jeanette Drew jdrew at cyllene.uwa.edu.au
Thu Nov 21 06:21:49 EST 1996


Hello friends,

As part of my PhD on ploidy and oncogene disturbances in endometrial cancer,
I am currently considering the viability of sorting tumours on the basis of
their ploidies with the intent of examining for allele loss in relevant
microsatellite regions.

A review of the literature, however, has yielded very little information on
this approach.  Generally, loss of heterozygosity is performed by
reverse-transcriptase/SSCP PCR methods on DNA extracted directly from fresh
or paraffin-embedded tissues.  

My enquiry is to anyone who may be doing similar studies as to what are some
of the issues I should be considering, and how viable this investigation may be.

Firstly, are there particular instrument requirements for undertaking
sorting of tumours with aneuploid populations in the order of 10-20%?

Will use of a BD FACSCalibur with Sort module be sufficient to separate
sufficient nuclei, or should I be looking at using a dedicated sorter?

Although I currently use PI to stain DNA, should I consider UV excititation
of DAPI for example if I have access to a UV laser on the sorter?

What laser configurations would be preferable, ie, type, power, etc.

Should sorting be decided on DNA-PI alone, or would dual parameter analysis
using anti-cytokeratin, for example, be preferable?

What numbers of cells are required post sorting for LoH studies?
(For other methods, 100 cells have been sufficient)

Finally, post sorting of diploid and aneuploid populations, are there other
investigations I could consider?

Any comments you have on these issues would be warmly welcomed.

Many thanks,

Jeanette.
_______________________________________________________________
Jeanette Drew
Department of Obstetrics & Gynaecology				   
University of Western Australia / King Edward Memorial Hospital
SUBIACO  WA  6009  AUSTRALIA				   	
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Tel: 61-9-340-2768					/     \	
Fax: 61-9-340-2636				  JD -> *_,-._/
Email: jdrew at cyllene.uwa.edu.au				     v
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